We have utilized molecular techniques to gain new insights into the behavior of hematopoietic stem and progenitor cells (HSPCs) in vivo. We have continued active development and utilization of lentiviral barcoding with high-diversity 31-35 base pair genetic barcodes utilized to label individual hematopoietic stem and progenitor cells to study in vivo hematopoiesis in the non-human primate model. Our collaborator Rong Lu first devised this very powerful approach and applied it to study murine hematopoiesis. We have now transplanted 20 macaques with barcoded autologous CD34+ cells, and have been able to track hematopoietic output from thousands of individual HSPCs over time (up to 5.5 years) and in multiple lineages in a quantitative and highly reproducible manner. We have already made a number of important and novel discoveries, including the lack of evidence for a common lymphoid progenitor producing T and B cells in primates, with no shared clonal derivation of B and T cells until late after transplant, and much earlier shared clonal derivation of myeloid and B cells. We have also for the first time discovered the unique lineage derivation and life history of the major fraction of natural killer (NK) cells. CD16+CD56- cytotoxic NK cells did not share barcodes with B, T or myeloid cells even 48 months post-transplant. In vitro and murine models have not previously been able to shed light on NK cell lineage relationships. We have continued to use barcoded cells from these macaques to further dissect in vivo NK cell ontogeny, and the process of ex vivo expansion of NK cells, highly relevant for adoptive cell therapy development, in collaboration with Dr. Rick Childs' group in NHLBI, Dr Miti Kaur's group at the Tulane Non-human Primate facility, and Dr Yenan Bryceson's group at the Karolinska Institut in Stockholm Sweden. We have mapped the clonally-unique barcodes to NK cells with an adaptive phenotype, linked in murine studies to NK memory, and in humans to putative NK adaptive responses to viruses such as CMV. Within NK cells with an adaptive phenotype, we find oligoclonal and massively expanded individual clones, with no contributions found in other lineages, and with waxing and waning patterns suggesting responses to specific environmental cues such as viral infection or reactivation. Our data provides the first direct demonstration of clonal NK responses, possibly explaining NK memory. With profound in vivo NK depletion, the same clones arise again, without recruitment from highly polyclonal HSPC. We hypothesized these clones are generated by a response to CMV, based on correlative data in human transplantation and blood donor studies, and are testing this via barcoded transplantation in CMV negative macaques. We continue to search for the precursor to these cells, tracking dominant clones in phenotypically purified samples from blood, bone marrow, lymph nodes, liver, and vaginal and intestinal lymphoid aggregates. We are also applying our methodology to understand the original of other classes of innate lymphoid cells (ILC), including ILC2 in the lung, and ILC3 in the gut. We validated our model suggesting that adaptive CD56dimCD16+ NK cells are produced independently of multipotent HSPC by taking advantage of two human disease models. In the human disorder PNH, acquired somatic mutations result in loss of GPI-linked proteins on HSPC and their progeny with clonal expansion of GPI- cells over time. We demonstrated that human CD56dimCD16+ NK cells from PNH patients are almost 100% GPI+ despite CD56bright NK cells, B cells and myeloid cells all being GPI-, similar to the pattern in T memory cells, and suggesting that adaptive NK cells share properties with T memory cells and can homotypically self-renew without ongoing production from HSPC (#2). We also demonstrated that human adaptive NK cells are retained patients with GATA2 deficiency syndrome, despite loss of HSPC, CD56bright NK cells, B cells and monocytes (#3). We have extended our analysis of the geographic segregation of individual HSPCs long term in specific marrow sites, confirming our prior findings in mice using LEGO imaging techniques now in the macaque model utilizing barcoding. We can directly demonstrate in situ production of B cells, CD56+ NK cells and myeloid cells in localized marrow niches, and surprisingly, we now have strong evidence for in situ marrow production of T cells (manuscript under review) We have followed the output of thousands of individual HSPC clones in several animals for up to 5.5 years, and demonstrate marked clonal stability of output from long-term repopulating clones producing myeloid, B cell and T cell lineages, along with CD56brightCD16- NK cells. We found no evidence for clonal succession in these young adult animals, directly refuting controversial murine data suggesting contributions from each clone lasting only several weeks. We have shown that myeloid vs lymphoid bias is common, and that this bias remains stable in individual clones over time (#1). We have recently completed a project comparing the clonal behavior of young versus aged HSPC, demonstrating marked differences in clonal patterns, specifically very delayed contributions from multilineage clones with persistence of unilineage contributions, in both myeloid, B and T cell lineages, in contrast to murine models suggesting accumulation of only myeloid-biased clones. In the oldest animal, we observed clonal dropout and clonal expansions, potentially providing a model of human CHIP associated with aging (clonal hematopoiesis of indeterminate prognosis). We have continued to analyze clonal patterns following engraftment of ex vivo expanded HSPC. We have derived methodologies allowing investigation of red cell and platelet lineages. Red cell clonal patterns match those of other myeloid lineages very closely. However, we have very interesting data regarding emergence of unique highly biased clones contributing to platelets in the presence of inflammation. We continue to collaboration with Rahul Sajita at NYU to apply single cell RNAseq to barcoded populations in order to further define ontogeny as well as identify rare precursor cell populations. We now have set up the DropSeq single cell methodology at NHLBI in collaboration with Andre Larochelle, and have evidence that we can retrieve both the barcode and the transcriptome from single cells. This approach offers many investigative opportunities, but also challenges in terms of data collection and analytics.